Systems and methods for using interleaving window widths in tandem mass spectrometry

ABSTRACT

Systems and methods are provided for analyzing a sample using overlapping measured mass selection window widths. A mass range of a sample is divided into two or more target mass selection window widths using a processor. The two or more target widths can have the same width or variable widths. A tandem mass spectrometer is instructed to perform two or more fragmentation scans across the mass range using the processor. Each fragmentation scan of the two or more fragmentation scans includes a measured mass selection window width. The two or more measured widths of the two or more fragmentation scans can have the same width or variable widths. At least two of the two or more measured mass selection window widths overlap. The overlap in measured mass selection window widths corresponds to at least one target mass selection window width.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.15/098,200, filed Apr. 13, 2016, which is a continuation of U.S. patentapplication Ser. No. 14/922,823, filed Oct. 26, 2015, now U.S. Pat. No.9,343,282, which is a continuation of U.S. patent application Ser. No.14/401,032, filed Nov. 13, 2014, filed as Application No.PCT/IB2013/000724 on Apr. 19, 2013, now U.S. Pat. No. 9,202,677, whichclaims the benefit of U.S. Provisional Patent Application No.61/649,199, filed May 18, 2012, the disclosures of which areincorporated by reference herein in their entireties.

INTRODUCTION

Both qualitative and quantitative information can be obtained from atandem mass spectrometer. In such an instrument a precursor ion isselected in a first mass analyzer, fragmented and the fragments analyzedin a second analyzer or in a second scan of the first analyzer. Thefragment ion spectrum can be used to identify the molecule and theintensity of one or more fragments can be used to quantitate the amountof the compound present in a sample.

Selected reaction monitoring (SRM) is a well-known example of this wherea precursor ion is selected, fragmented, and passed to a second analyzerwhich is set to transmit a single ion. A response is generated when aprecursor of the selected mass fragments to give an ion of the selectedfragment mass, and this output signal can be used for quantitation. Theinstrument may be set to measure several fragment ions for confirmationpurposes or several precursor-fragment combinations to quantitatedifferent compounds.

The sensitivity and specificity of the analysis are affected by thewidth of the mass window selected in the first mass analysis step. Widewindows transmit more ions giving increased sensitivity, but may alsoallow ions of different mass to pass; if the latter give fragments atthe same mass as the target compound interference will occur and theaccuracy will be compromised.

In some mass spectrometers the second mass analyzer can be operated athigh resolution, allowing the fragment ion window to be narrow so thatthe specificity can to a large degree be recovered. These instrumentsmay also detect all fragments so they are inherently detecting differentfragments. With such an instrument it is feasible to use a wide windowto maximize sensitivity.

These recently developed high-resolution and high-throughput instrumentsallow a mass range to be accurately scanned within a time interval usingmultiple scans with adjacent or overlapping mass window widths. Thecollection of each spectrum at each time interval of the separation is acollection of spectra for the entire mass range. One exemplary methodfor using windowed mass spectrometry scans to scan an entire mass rangeis called sequential windowed acquisition (SWATH).

Currently a SWATH user has to balance the number of SWATH experiments,the accumulation time, and also the number of data points across a peak.For example, if the user tries to use narrow mass window widths across amass range the result may be that there is not enough sensitivity or thecycle time is too large to provide sufficient data points across a peak.

BRIEF DESCRIPTION OF THE DRAWINGS

The skilled artisan will understand that the drawings, described below,are for illustration purposes only. The drawings are not intended tolimit the scope of the present teachings in any way.

FIG. 1 is a block diagram that illustrates a computer system, upon whichembodiments of the present teachings may be implemented.

FIG. 2 is an illustration of the mass coverage of overlapping measuredmass selection window widths of uniform length that are used to scantarget mass selection window widths of uniform length with the samenumber of overlapping measured mass selection window widths, inaccordance with various embodiments.

FIG. 3 is an illustration of the mass coverage of overlapping measuredmass selection window widths of variable length that are used to scantarget mass selection window widths of variable length with the samenumber of overlapping measured mass selection window widths, inaccordance with various embodiments.

FIG. 4 is an illustration of the mass coverage of overlapping measuredmass selection window widths of uniform length that are used to scantarget mass selection window widths of uniform length with a variablenumber of overlapping measured mass selection window widths, inaccordance with various embodiments.

FIG. 5 is a schematic diagram showing a system for analyzing a sampleusing overlapping measured mass selection window widths, in accordancewith various embodiments.

FIG. 6 is an exemplary flowchart showing a method for analyzing a sampleusing overlapping measured mass selection window widths, in accordancewith various embodiments.

FIG. 7 is a schematic diagram of a system that includes one or moredistinct software modules that performs a method for analyzing a sampleusing overlapping measured mass selection window widths, in accordancewith various embodiments.

Before one or more embodiments of the present teachings are described indetail, one skilled in the art will appreciate that the presentteachings are not limited in their application to the details ofconstruction, the arrangements of components, and the arrangement ofsteps set forth in the following detailed description or illustrated inthe drawings. Also, it is to be understood that the phraseology andterminology used herein is for the purpose of description and should notbe regarded as limiting.

DESCRIPTION OF VARIOUS EMBODIMENTS

Computer-Implemented System

FIG. 1 is a block diagram that illustrates a computer system 100, uponwhich embodiments of the present teachings may be implemented. Computersystem 100 includes a bus 102 or other communication mechanism forcommunicating information, and a processor 104 coupled with bus 102 forprocessing information. Computer system 100 also includes a memory 106,which can be a random access memory (RAM) or other dynamic storagedevice, coupled to bus 102 for storing instructions to be executed byprocessor 104. Memory 106 also may be used for storing temporaryvariables or other intermediate information during execution ofinstructions to be executed by processor 104. Computer system 100further includes a read only memory (ROM) 108 or other static storagedevice coupled to bus 102 for storing static information andinstructions for processor 104. A storage device 110, such as a magneticdisk or optical disk, is provided and coupled to bus 102 for storinginformation and instructions.

Computer system 100 may be coupled via bus 102 to a display 112, such asa cathode ray tube (CRT) or liquid crystal display (LCD), for displayinginformation to a computer user. An input device 114, includingalphanumeric and other keys, is coupled to bus 102 for communicatinginformation and command selections to processor 104. Another type ofuser input device is cursor control 116, such as a mouse, a trackball orcursor direction keys for communicating direction information andcommand selections to processor 104 and for controlling cursor movementon display 112. This input device typically has two degrees of freedomin two axes, a first axis (i.e., x) and a second axis (i.e., y), thatallows the device to specify positions in a plane.

A computer system 100 can perform the present teachings. Consistent withcertain implementations of the present teachings, results are providedby computer system 100 in response to processor 104 executing one ormore sequences of one or more instructions contained in memory 106. Suchinstructions may be read into memory 106 from another computer-readablemedium, such as storage device 110. Execution of the sequences ofinstructions contained in memory 106 causes processor 104 to perform theprocess described herein. Alternatively hard-wired circuitry may be usedin place of or in combination with software instructions to implementthe present teachings. Thus implementations of the present teachings arenot limited to any specific combination of hardware circuitry andsoftware.

The term “computer-readable medium” as used herein refers to any mediathat participates in providing instructions to processor 104 forexecution. Such a medium may take many forms, including but not limitedto, non-volatile media, volatile media, and transmission media.Non-volatile media includes, for example, optical or magnetic disks,such as storage device 110. Volatile media includes dynamic memory, suchas memory 106. Transmission media includes coaxial cables, copper wire,and fiber optics, including the wires that comprise bus 102.

Common forms of computer-readable media include, for example, a floppydisk, a flexible disk, hard disk, magnetic tape, or any other magneticmedium, a CD-ROM, digital video disc (DVD), a Blu-ray Disc, any otheroptical medium, a thumb drive, a memory card, a RAM, PROM, and EPROM, aFLASH-EPROM, any other memory chip or cartridge, or any other tangiblemedium from which a computer can read.

Various forms of computer readable media may be involved in carrying oneor more sequences of one or more instructions to processor 104 forexecution. For example, the instructions may initially be carried on themagnetic disk of a remote computer. The remote computer can load theinstructions into its dynamic memory and send the instructions over atelephone line using a modem. A modem local to computer system 100 canreceive the data on the telephone line and use an infra-red transmitterto convert the data to an infra-red signal. An infra-red detectorcoupled to bus 102 can receive the data carried in the infra-red signaland place the data on bus 102. Bus 102 carries the data to memory 106,from which processor 104 retrieves and executes the instructions. Theinstructions received by memory 106 may optionally be stored on storagedevice 110 either before or after execution by processor 104.

In accordance with various embodiments, instructions configured to beexecuted by a processor to perform a method are stored on acomputer-readable medium. The computer-readable medium can be a devicethat stores digital information. For example, a computer-readable mediumincludes a compact disc read-only memory (CD-ROM) as is known in the artfor storing software. The computer-readable medium is accessed by aprocessor suitable for executing instructions configured to be executed.

The following descriptions of various implementations of the presentteachings have been presented for purposes of illustration anddescription. It is not exhaustive and does not limit the presentteachings to the precise form disclosed. Modifications and variationsare possible in light of the above teachings or may be acquired frompracticing of the present teachings. Additionally, the describedimplementation includes software but the present teachings may beimplemented as a combination of hardware and software or in hardwarealone. The present teachings may be implemented with bothobject-oriented and non-object-oriented programming systems.

Systems and Methods of Data Processing

As described above, one exemplary method for using windowed massspectrometry scans to scan an entire mass range is called sequentialwindowed acquisition (SWATH). However, a SWATH user has to balance thenumber of SWATH experiments, the accumulation time, and also the numberof data points across a peak.

For example, the SWATH technique provides a method to generate production spectra for all species detectable in a liquid chromatographycoupled mass spectrometry (LCMS) analysis. This is achieved by using awide precursor selection window that is stepped across the mass range ofinterest. An exemplary precursor selection window is 25 amu, but othervalues and even variable widths are possible. Choosing window widths andaccumulation times is a balancing act with several considerations:

First, to retain liquid chromatography (LC) peak fidelity, ideally eightto ten data points per peak are needed. This defines the time that canbe spent on each cycle.

Due to the nature of the method, maximum coverage of data occurs whenthe complete mass range is scanned. This results in a number of steps(windows) being required to cover the mass range.

To maintain a high degree of selectivity, the windows are required to beas narrow as possible, resulting in a few precursors per window. Thisprovides less chance of fragment ion interference. This also increasesthe number of windows and reduces the time required for each window.

Finally, to maximize both dynamic range and sensitivity, a maximumaccumulation time is required for each window.

In various embodiments, systems and methods decrease the widths of thewindows in a sequential windowed acquisition while increasing the timethat is spent on each window. Windows are overlapped in order to analyzeeach region more than once and to extract the required informationpost-acquisition.

FIG. 2 is an illustration 200 of the mass coverage of overlappingmeasured mass selection window widths of uniform length that are used toscan target mass selection window widths of uniform length with the samenumber of overlapping measured mass selection window widths, inaccordance with various embodiments. The mass range 210 is spanned usingtarget mass selection window widths B, C, and D, for example. The targettime per target mass selection window widths is t. Using wider measuredmass selection window widths Scan 1, Scan 2, Scan 3, and Scan 4 withoverlaps that correspond to the target mass selection window widths B,C, and D, the equivalent information is extracted in less time. Forexample, Equation 1 shows that by covering mass window C twice, thecorrect coverage is obtained by analyzing each window for half of thetime t.2C=Scan 2+Scan 3−Scan 1−Scan 4  (1)

Illustration 200 shows that measured mass selection window widths areoffset by 50% of the target mass selection window width so that theeffective scanning window is one half of the width actually used. InFIG. 2, each target mass selection window width is overlapped by twomeasured mass selection window widths. More overlap can also be providedwith more or wider measured mass selection window widths, furtherreducing accumulation times to obtain narrower effective widths. Moreoverlap simply increases the number of scans that are summed andsubtracted to generate the desired result. This method allows widermeasured windows to be used while maintaining the benefit of narrowtarget windows and less time to be spent on each measured window.

In FIG. 2, both the measured mass selection window widths and the targetmass selection window widths have uniform widths. In variousembodiments, the measured mass selection window widths, the target massselection window widths, or both widths can be variable. In FIG. 2, thenumber of overlaps of measured mass selection window widthscorresponding to each target mass selection window width is uniform. Invarious embodiments, the number of overlaps of measured mass selectionwindow widths that correspond to target mass selection window widths canbe variable. In various embodiments, any combination of measured ortarget window widths or number of overlaps of measured mass selectionwindow widths can be used.

FIG. 3 is an illustration 300 of the mass coverage of overlappingmeasured mass selection window widths of variable length that are usedto scan target mass selection window widths of variable length with thesame number of overlapping measured mass selection window widths, inaccordance with various embodiments. In FIG. 3, target mass selectionwindow widths B and C have different widths, and measured mass selectionwindow widths Scan 1 and Scan 2 have different widths, for example.

FIG. 4 is an illustration 400 of the mass coverage of overlappingmeasured mass selection window widths of uniform length that are used toscan target mass selection window widths of uniform length with avariable number of overlapping measured mass selection window widths, inaccordance with various embodiments. In FIG. 4, the number of overlapsof measured mass selection window widths that correspond to target massselection window widths B and C are different, for example.

Tandem Mass Spectrometry System

FIG. 5 is a schematic diagram showing a system 500 for analyzing asample using overlapping measured mass selection window widths, inaccordance with various embodiments. System 500 includes tandem massspectrometer 510 and processor 520. Processor 520 can be, but is notlimited to, a computer, microprocessor, or any device capable of sendingand receiving control signals and data from mass spectrometer 510 andprocessing data.

Tandem mass spectrometer 510 can include one or more physical massanalyzers that perform two or more mass analyses. A mass analyzer of atandem mass spectrometer can include, but is not limited to, atime-of-flight (TOF), quadrupole, an ion trap, a linear ion trap, anorbitrap, or a Fourier transform mass analyzer. Tandem mass spectrometer510 can also include a separation device (not shown). The separationdevice can perform a separation technique that includes, but is notlimited to, liquid chromatography, gas chromatography, capillaryelectrophoresis, or ion mobility. Tandem mass spectrometer 510 caninclude separating mass spectrometry stages or steps in space or time,respectively.

Tandem mass spectrometer 510 includes a mass analyzer that allowsoverlapping measured mass selection window widths.

Processor 520 is in communication with tandem mass spectrometer 510.Processor 520 divides a mass range of a sample into two or more targetmass selection window widths. The two or more target mass selectionwindow widths are based on a minimum selectivity requirement. The two ormore target mass selection window widths can have the same width orvariable widths.

Processor 520 instructs tandem mass spectrometer 510 to perform two ormore fragmentation scans across the mass range. Each fragmentation scanof the two or more fragmentation scans has a measured mass selectionwindow width. The two or more measured mass selection window widths ofthe two or more fragmentation scans can have the same width or variablewidths. At least two of the two or more measured mass selection windowwidths overlap. The overlap in measured mass selection window widthscorresponds to at least one target mass selection window width of thetwo or more target mass selection window widths.

In various embodiments, each target mass selection window width of thetwo or more target mass selection window widths corresponds tooverlapped measured mass selection window widths. The number of measuredmass selection window widths corresponding to target mass selectionwindow widths can be the same or variable across the two or more targetmass selection window widths.

In various embodiments, processor 520 extracts information about atleast one target mass selection window width by combining informationfrom corresponding overlapped measured mass selection window widths ofat least two fragmentation scans. The information is combined using amathematical or logical operation, for example.

Tandem Mass Spectrometry Method

FIG. 6 is an exemplary flowchart showing a method 600 for analyzing asample using overlapping measured mass selection window widths, inaccordance with various embodiments.

In step 610 of method 600, a mass range of a sample is divided into twoor more target mass selection window widths using a processor.

In step 620, a tandem mass spectrometer is instructed to perform two ormore fragmentation scans across the mass range using the processor. Eachfragmentation scan of the two or more fragmentation scans includes ameasured mass selection window width. An overlap in measured massselection window widths of at least two fragmentation scans of the twoor more fragmentation scans corresponds to at least one target massselection window width of the two or more target mass selection windowwidths.

Tandem Mass Spectrometry Computer Program Product

In various embodiments, a computer program product includes a tangiblecomputer-readable storage medium whose contents include a program withinstructions being executed on a processor so as to perform a method foranalyzing a sample using overlapping measured mass selection windowwidths. This method is performed by a system that includes one or moredistinct software modules.

FIG. 7 is a schematic diagram of a system 700 that includes one or moredistinct software modules that performs a method for analyzing a sampleusing overlapping measured mass selection window widths, in accordancewith various embodiments. System 700 includes an analysis module 710 anda fragmentation scan module 720.

Analysis module 710 divides a mass range of a sample into two or moretarget mass selection window widths. Fragmentation scan module 720instructs a tandem mass spectrometer to perform two or morefragmentation scans across the mass range. Each fragmentation scan ofthe two or more fragmentation scans includes a measured mass selectionwindow width. An overlap in measured mass selection window widths of atleast two fragmentation scans of the two or more fragmentation scanscorresponds to at least one target mass selection window width of thetwo or more target mass selection window widths.

While the present teachings are described in conjunction with variousembodiments, it is not intended that the present teachings be limited tosuch embodiments. On the contrary, the present teachings encompassvarious alternatives, modifications, and equivalents, as will beappreciated by those of skill in the art.

Further, in describing various embodiments, the specification may havepresented a method and/or process as a particular sequence of steps.However, to the extent that the method or process does not rely on theparticular order of steps set forth herein, the method or process shouldnot be limited to the particular sequence of steps described. As one ofordinary skill in the art would appreciate, other sequences of steps maybe possible. Therefore, the particular order of the steps set forth inthe specification should not be construed as limitations on the claims.In addition, the claims directed to the method and/or process should notbe limited to the performance of their steps in the order written, andone skilled in the art can readily appreciate that the sequences may bevaried and still remain within the spirit and scope of the variousembodiments.

What is claimed is:
 1. A system for analyzing a sample using overlappingmeasured mass selection window widths, comprising: a tandem massspectrometer that includes a mass analyzer that allows overlappingmeasured mass selection window widths; and a controller in communicationwith the tandem mass spectrometer that is configured to divide a massrange of a sample into two or more target mass selection window widths,and configured to instruct the tandem mass spectrometer to fragment eachregion of the mass range at least twice using overlapping measured massselection window widths, wherein an overlap in at least two measuredmass selection window widths corresponds to at least one target massselection window width.
 2. The system of claim 1, wherein the two ormore target mass selection window widths have the same width.
 3. Thesystem of claim 1, wherein the two or more target mass selection windowwidths have variable widths.
 4. The system of claim 1, wherein the twoor more measured mass selection window widths have the same width. 5.The system of claim 1, wherein the two or more measured mass selectionwindow widths have variable widths.
 6. The system of claim 1, whereineach target mass selection window width of the two or more target massselection window widths corresponds to overlapped measured massselection window widths that include the same number of measured massselection window widths.
 7. The system of claim 1, wherein each targetmass selection window width of the two or more target mass selectionwindow widths corresponds to overlapped measured mass selection windowwidths that include a variable number of measured mass selection windowwidths.
 8. The system of claim 1, wherein the processor further extractsinformation about the at least one target mass selection window width bycombining information from the measured mass selection window widths,wherein equivalent information is extracted in less time.
 9. A systemfor analyzing a sample using overlapping measured mass selection windowwidths, comprising: a tandem mass spectrometer that includes a massanalyzer that allows overlapping measured mass selection window widths;and a processor in communication with the tandem mass spectrometer, theprocessor being coupled to a storage medium encoded to perform stepscomprising: dividing a mass range of a sample into two or more targetmass selection window widths, and instructing the tandem massspectrometer to fragment each region of the mass range at least twiceusing overlapping measured mass selection window widths, wherein anoverlap in at least two measured mass selection window widthscorresponds to at least one target mass selection window width.
 10. Thesystem of claim 9, wherein the two or more target mass selection windowwidths have the same width.
 11. The system of claim 9, wherein the twoor more target mass selection window widths have variable widths. 12.The system of claim 9, wherein the two or more measured mass selectionwindow widths have the same width.
 13. The system of claim 9, whereinthe two or more measured mass selection window widths have variablewidths.
 14. The system of claim 9, wherein each target mass selectionwindow width of the two or more target mass selection window widthscorresponds to overlapped measured mass selection window widths thatinclude the same number of measured mass selection window widths. 15.The system of claim 9, wherein each target mass selection window widthof the two or more target mass selection window widths corresponds tooverlapped measured mass selection window widths that include a variablenumber of measured mass selection window widths.
 16. The system of claim9, wherein the processor further extracts information about the at leastone target mass selection window width by combining information from themeasured mass selection window widths, wherein equivalent information isextracted in less time.
 17. A method for analyzing a sample usingoverlapping measured mass selection window widths, comprising: dividinga mass range of a sample into two or more target mass selection windowwidths using a processor, and instructing a tandem mass spectrometer tofragment each region of the mass range at least twice using overlappingmeasured mass selection window widths using the processor, wherein anoverlap in at least two measured mass selection window widthscorresponds to at least one target mass selection window width.
 18. Themethod of claim 17, wherein the two or more target mass selection windowwidths have the same width.
 19. The method of claim 17, wherein the twoor more target mass selection window widths have variable widths. 20.The method of claim 17, wherein the two or more measured mass selectionwindow widths have the same width.